High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients

Abstract

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC).

Patients and methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival.

Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls.

Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

Keywords: KRAS; circulating tumor DNA; exosome; liquid biopsy; pancreatic cancer.

Figure 1.

Liquid biopsy Kaplan–Meier curves. (A) Stratification of exoKRAS at a mutant allele frequency of 1% was associated with disease free survival in patients with localized disease who were treatment naïve at the time of blood draw (n = 13) with a median survival of 441 days compared to 127 days (P = 0.031). Two treatment naïve patients with no KRAS mutant droplets were excluded from this survival analysis to account for the possibility that they have a KRAS mutation that is not a target of the KRAS multiplex ddPCR kit used. (B and C) Exosome concentration of 5 × 10⁹ per ml of plasma was associated with overall survival in treatment naïve blood draws in patients with (B) localized disease (n = 15, median survival 616 versus 233 days, P = 0.048) and (C) metastatic disease (n = 12, median survival 479 versus 97 days, P = 0.015).